WISP1 and TLR4 Signaling in ventilator-induced lung injury (VILI)

描述（由申请人提供）：尽管急性肺损伤结局的改善主要归因于肺保护性机械通气策略，但呼吸机诱导的肺损伤（VILI）仍然是危重患者发病率和死亡率的重要因素。使用无偏的全基因组关联研究，我们（Li等人，Am J Resp Cell Mol Biol 2012）鉴定了鼠VILI中的WNT 1诱导型信号传导途径蛋白1（WISP 1）。此外，我们和其他人已经确定，通过TLR 4的先天免疫信号传导在VILI的发病机制中起着关键作用，并且牵张诱导的WISP 1表达及其促炎作用是TLR 4依赖的。因此，我们建议：具体目标1。确定机械拉伸与呼吸道上皮WISP 1生物合成和VILI相关的分子途径。我们将：a）使用药理学和遗传学方法来剖析非经典Wnt信号传导途径在循环拉伸培养的鼠呼吸道上皮中WISP 1生物合成中的贡献; B）使用生物化学决定簇来关联HIV后小鼠完整肺中的非经典途径（12 ml/kg x 6 h）;和c）通过野生型HTV后肺泡毛细血管通透性的变化确定上皮来源的WISP 1在VILI中的作用，WISP 1-/-和在用shRNA的肺内慢病毒递送原位沉默肺泡巨噬细胞中的WISP 1之后（并且因此留下上皮来源的WISP 1作为唯一来源）。具体目标2。为了确定WISP 1在将机械应激反应传递给先天免疫系统导致VILI中的作用：我们将通过比较HIV对野生型、全身TLR 4和骨髓细胞特异性TLR 4缺失小鼠的作用来确定巨噬细胞（和嗜中性粒细胞）TLR 4信号传导在VILI中的贡献（Lyz-TLR 4; Nace等人，Hepatology 2013）。具体目标3。确定WISP 1作为辅助分子通过TLR 4将气道上皮的机械拉伸应力转导为巨噬细胞的促炎表型的分子决定因素。我们将使用腹腔巨噬细胞确定WISP 1促炎（例如TNFα; NFκB）作用的必要组分，并确认野生型和CD 14以及β 3和β 5缺失小鼠的原代小鼠肺泡巨噬细胞培养物（数量有限）中积累的信息。然后，我们将通过对比HIV对β 3和β 5缺失小鼠的作用，用和不用i.t.注射WISP 1。总的来说，这些研究将提供新的见解的作用，基质细胞蛋白，WISP 1，和Wnt通路，在转导的机械应力对呼吸道上皮细胞的影响，先天免疫系统和TLR 4激活肺泡巨噬细胞的作用在VILI的发病机制。这种机制的见解可能会导致生物标志物，在预防或减轻VILI的治疗靶点和进一步了解VILI的易感性的遗传决定因素。

DESCRIPTION (provided by applicant): Although much of the improvement in outcomes from acute lung injury has been ascribed to lung-protective mechanical ventilation strategies, ventilator-induced lung injury (VILI) remains an important element of morbidity and mortality in the critically ill patient. Using an unbiased genome-wide association study, we (Li et al, Am J Resp Cell Mol Biol 2012) identified a WNT1-inducible signaling pathway protein 1 (WISP1) in murine VILI. Moreover, we and others have identified that innate immune signaling via TLR4 plays a critical role in the pathogenesis of VILI and that stretch-induced WISP1 expression and its pro-inflammatory effect were TLR4- dependent. Accordingly, we propose: Specific Aim 1. To determine the molecular pathway by which mechanical stretch is coupled to respiratory epithelial WISP1 biosynthesis and VILI. We will: a) use pharmacological and genetic approaches to dissect contribution of non-canonical Wnt signaling pathway in WISP1 biosynthesis in cyclic stretched cultured murine respiratory epithelium; b) use biochemical determinants to associate non-canonical pathway in intact lung of mice after HTV (12 ml/kg x 6h); and c) determine the contribution of epithelial derived WISP1 in VILI by changes in alveolar capillary permeability after HTV in wildtype, WISP1 -/- and after silencing WISP1 in alveolar macrophages in situ with intratracheal lentiviral delivery of shRNA (and hence leaving epithelial derived WISP1 as sole source). Specific Aim 2. To determine the role of WISP1 in communicating mechanical stress responses to innate immune system leading to VILI: We will determine the contribution of macrophage (and neutrophil) TLR4 signaling in VILI by comparing the effect of HTV on wildtype, whole body TLR4 and myeloid-cell-specific TLR4 null mice (Lyz-TLR4; Nace et al, Hepatology 2013). Specific Aim 3. To determine the molecular determinants by which WISP1 acts as an accessory molecule transducing stress of mechanical stretch in airway epithelium to pro-inflammatory phenotype of macrophages via TLR4. We will define requisite components for proinflammatory (e.g. TNFα; NFκB) effect of WISP1 using peritoneal macrophages and confirm accrued information in primary murine alveolar macrophage cultures (that are limiting in number) from wildtype and CD14 and ß3 and ß5 null mice. We will then determine the obligatory roles of ß3 and ß5 in WISP1 mediated VILI by contrasting the effect of HTV on ß3 and ß5 null mice, with and without i.t. injection of WISP1. Collectively, these studies will provide novel insight into the role of matricellular protein, WISP1, and the Wnt pathway, in transducing the effect of mechanical stress on respiratory epithelium to innate immune system and role of TLR4 activation of alveolar macrophages in pathogenesis of VILI. Such mechanistic insight may lead to biomarkers, therapeutic targets in prevention or mitigation of VILI and further understanding of genetic determinants of susceptibility to VILI.